EP1751268B1 - Mikrofluidische vorrichtung und leukozyentantigen-vermittelter mikrofluidischer assay - Google Patents

Mikrofluidische vorrichtung und leukozyentantigen-vermittelter mikrofluidischer assay Download PDF

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EP1751268B1
EP1751268B1 EP05856714.0A EP05856714A EP1751268B1 EP 1751268 B1 EP1751268 B1 EP 1751268B1 EP 05856714 A EP05856714 A EP 05856714A EP 1751268 B1 EP1751268 B1 EP 1751268B1
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microchamber
observation
sample
antigen
leucocyte
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EP1751268A2 (de
EP1751268A4 (de
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Rodolfo Ramiro Rodriguez
Douglas James Darr
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Advanced Animal Diagnostics Inc
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Advanced Animal Diagnostics Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/28—Constructional details, e.g. recesses, hinges disposable or single use
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors

Definitions

  • the present invention relates to an assay method and a microfluidic device for analyzing a subjects' body fluids containing leucocytes to determine if the subject has been previously exposed to the antigen.
  • the invention relates to an assay for observation, qualification and quantification of leucocyte measurement factors in a leucocyte containing fluid sample from a subject by exposure to a predetermined antigen in a novel microfluidic device in order to determine if there has been a prior exposure to the antigen.
  • Antigens can be disease pathogens, allergy agents, toxins, infectious agents and the like. These days, because of the threat of bio-terrorism, not only are physicians concerned with normal exposure of their patients to various pathogens, the possibility of a civilian or military population being exposed to a terrorist act such as the anthrax events of late has created a need for tests which are simple, can be performed almost anywhere, are accurate and fast to determine if there has been a first prior exposure to a predetermined antigen in order to determine what if any method of treatment to use on the subject. In addition to the above, prior exposure to an antigen necessarily results after vaccination for infectious diseases. It is often desirable for a physician to determine that a vaccination has been successful in immunizing a subject or that after an extended time period that immunization from a subjects' prior vaccination remains above an acceptable level.
  • Antibodies are produced by certain lymphocytes, the B-lymphocytes, and are one of the body's defense mechanism against antigens in the blood stream and other body fluids containing leucocyte.
  • the presence of circulating antibodies is an indicator that the subject is or has been actively fighting against such infectious agents as viral or bacterial antigens.
  • a plethora of tests have been developed to directly and indirectly measure circulating antibodies from an ex vivo blood sample. More common methods include latex agglutination, enzyme linked immuno substrate assay, radioimmunoassay, radio immuno sorbant assay, indirect immuno fluorescence and the like. These antibody assays primarily rely on the production of antibody by B-lymphocytes and the measurement of their presence in sufficient quantities in the blood sample.
  • T-lymphocytes especially memory T-lymphocytes
  • T-lymphocytes are known to quickly undergo changes upon second antigen challenge. These changes are collectively referred to as measurement factors.
  • Current lymphocyte testing relies on measurements of morphological factors and/or biochemical factors as an indication of activation by an antigen. Some of the known factors are listed in US patent 5,480,778 . These types of tests also have several difficulties.
  • the patent does not disclose comparing samples of the same cell types for differences in measurement factors, other than quantity of cells information it doesn't discuss any measurement factor observations at all and does not disclose use of this technique to identify cells which have been exposed to an antigen.
  • the patent further does not describe control methods and does not describe use in diagnosis of disease states other than inferring it can perform cell counts.
  • the apparatus for scanning a fluid sample (for example by fluorescence analysis using computer pixel modeling) is known in the art.
  • Commercial fluorescence readers are readily available and can be programmed by one skilled in the art to read and analyze fluorescence data as needed.
  • WO94/10576 describes a method and apparatus for measuring immunoactivity comprising exposure of a leucocyte population to a potential allergen, preferably on a membrane material which is adapted to selectively retain the leucocyte component of a blood sample and the subsequent stressing of the leucocyte population to bring about release of an agent generated by leucocytes as a result of their involvement in an immunogenic reaction; and finally the detection of said agent using a colourimetric agent and measuring means.
  • US5116731 describes a one-step process for the detection of the presence of an allergy and for the specific detection of the allergen responsible for the allergy, in which the leukocytes of a sample to be investigated are incubated with an allergen or with another stimulation factor in an aqueous medium together with a chromogenic protease substrate and calcium ions, the liberated protease is reacted with the chromogen and the resulting chromophor is determined.
  • the protease activity is measured kinetically after an incubation period by the increase of the chromophor concentration.
  • a device for carrying out this process which consists of a microtiter plate with a plurality of different reagents arranged in rows.
  • US5360719 describes a method in which a patient's blood sample is incubated with an antigen and tested for lymphocyte response, i.e. an activation of lymphocytes and/or a conversion of lymphocytes to lymphoblasts, which indicates prior exposure of the patient to the antigen.
  • lymphocyte response i.e. an activation of lymphocytes and/or a conversion of lymphocytes to lymphoblasts
  • a positive response indicates the presence of prior exposure to prior diseases or clinical conditions such as parasitic diseases, tuberculosis, salmonellosis, gonorrhea, fungal infections, rickettsial infections, Lyme disease or allergens.
  • Whole blood from the patient is incubated with the antigen of the disease or condition for which the patient is being tested.
  • a fluorescent dye or colorant which has an affinity for a discriminant characteristic of the activated lymphocytes or lymphoblasts, such as: intracellular calcium; surface activation antigens such as transferrin receptor; HLA-Dr; Leu-23; and the like.
  • the incubated blood is then drawn into a transparent tube containing a float which concentrates the buffy coat constituent layers upon centrifugation of the blood sample.
  • the concentrated lymphocyte layer is then examined for fluorescence or coloration which is indicative of the presence of the activated lymphocytes or lymphoblasts, and their concentration.
  • the fluorescence or coloration can be qualified and/or quantified by a reader instrument.
  • the method described is a leucocyte antigen mediated microfluidic assay for testing a leucocyte containing fluid from a subject for prior exposure to a predetermined antigen which would have caused the leucocyte to become pre-sensitized comprising:
  • the device as described is a microfluidic device for use in performing a leucocyte-antigen mediated microfluidic assay in a leucocyte containing fluid sample from a subject comprising:
  • the colorant is not cytotoxic and the first reaction microchamber and the first observation microchamber are the same microchamber; and the second reaction microchamber and the second observation microchamber are the same microchamber.
  • first and second sample addition microchambers are the same microchamber.
  • the described subject matter also relates to a kit of parts comprising both the microfluidic device as well as the apparatus which is capable of reading the microfluidic device observation microchambers and comparing the two performed tests to determine the status of antigen exposure of leucocytes in the fluid of a test subject.
  • the method of the invention as claimed is a leucocyte antigen mediated microfluidic assay method for testing a leucocyte-containing body fluid from a subject for prior exposure to a predetermined antigen which would have caused the leucocyte to become pre-sensitized to the antigen, the method comprising:
  • the device of the invention as claimed is a microfluidic device for use in performing the leucocyte-antigen mediated assay method of the invention as claimed, the device comprising:
  • the kit of the invention as claimed is a kit of parts comprising a microfluidic device which is a device of the invention as claimed and an apparatus capable of performing an optical multifield scan on the observation microchambers in the microfluidic device.
  • the present invention relates to a microfluidic device and a leucocyte antigen mediated microfluidic assay (LAMMA) for determination if a subject has been previously exposed to a predetermined antigen by testing the subjects' leucocyte containing body fluid sample to see if the leucocytes are presensitized to the predetermined antigen.
  • LAMMA leucocyte antigen mediated microfluidic assay
  • leucocyte containing body fluid such as whole blood or other leucocyte containing body fluid such as milk, lymph fluid, spinal fluid or the like
  • the tests are compared for difference in leucocyte measurement factors and those differences determine if the subject has been previously exposed to the predetermined antigen i.e. if the leucocyte is presensitized.
  • leucocyte response which is a direct indicator of level a vaccination protection, can be determined by such measurements using the present invention.
  • Vaccination response can be used after initial vaccination to determine if titer levels are sufficient for protection or after an extended period of time to determine if a previous vaccination is still providing sufficient levels of protection. This can be accomplished by observing the number of reacting cells that exhibit a measurement factor, as well as the plus or minus intensity of the difference between the 2 tests.
  • subject is meant herein to be any animal especially birds, mammals and especially humans, that have whole blood circulating leucocytes or other body fluids containing leucocyte, especially T-lymphocytes, and especially memory T-lymphocytes that produce a measurement factor change after exposure to an antigen.
  • the antigen exposure of the subject is either from an environmental source such as infection or disease caught from another animal or the environment, vector transmitted antigens, zoonotic transmitted antigens or in the case of bio-terrorism an antigen introduced to the subject population by artificial means such as disposal in water sources, release into the environment, insertion into the food supply, use of mail systems or other mechanical means and the like.
  • leucocyte and "presensitized leucocyte” is meant any of the sub categories of leucocytes that have been exposed previously to an antigen or would participate in reaction to re-exposure to that antigen that will then exhibit a one or more measurement factors upon a subsequent exposure to the same antigen. While previous assays have limited testing to a particular leucocyte subclass such as T lymphocytes it is now possible to measure changes in all leucocyte that exhibit a measurement factor regardless if it is directly or indirectly reacting with an antigen. Cells include not only all lymphocytes but neutrophils and other white blood cells. A distinct advantage of this assay occurs when assaying for neutrophil measurement factors in that they are much more plentiful than other leucocytes and therefore smaller samples and less search time and effort is required to find them in a sample.
  • Anticoagulated whole blood as claimed herein can be produced by adding anticoagulants to whole blood either prior to the addition of whole blood to the sample microchamber or anticoagulant can be added to the sample microchamber either before or after addition of the blood to the microchamber.
  • the LAMMA and microfluidic device of the present invention requires only microvolumes of body fluid. In general, those amounts would be in microliter amounts and in general about 100 microliters or less, about a single drop of blood or other body fluid, for each microchamber. In the case where a single microchamber is used as the first and second sample microchamber, it is possible to start with a single drop of body fluid. Single drops of body fluid range from about 10 microliters to about 60 microliters. Whole blood and other fluids can be taken from either a previous larger sample or taken directly from the subject for the method of the invention, for example, a drop of blood obtained by lancet. Placement can then be made into the microchamber by either direct placement from the lanced body part or by mechanical means such as by dropper or pipette.
  • fluid communication between microchambers means that channels are produced of appropriate size for the microvolumes of leucocyte containing body fluid being tested.
  • Small capillary type channels in the device having a diameter of about 30 micrometers to about 2 millimeters, more or less are appropriate. Where a fluid contains larger molecules than in whole blood, for example the large proteins in milk, appropriate larger channels can be used.
  • a means for moving the fluid In order to move the fluid between chambers a means for moving the fluid must be used.
  • One particular method of moving body fluids between microchambers is capillary action that will move fluid almost immediately between microchambers. Where slightly larger channels are used between microchambers a micropump of appropriate size can be used.
  • a small valve or other blocking device can be inserted in the following channel to prevent capillary action or other transportation means from occurring prior to the end of the reaction time.
  • sample microchamber of the invention is designed to accept the whole blood or other leucocyte containing body fluid sample of the subject. It can be two separate microchambers, one for each of the two tests performed by the method or it can be a single microchamber with dual fluid communication means such as channels, one for each test provided, such that roughly equal portions of the fluid sample are delivered for each of the two reaction microchambers.
  • the microchambers are design to accept the microvolumes of fluid from the subject, so therefore their volume is roughly equal to or slightly larger than the anticipated test sample of body fluid.
  • reaction microchamber is in direct fluid communication with the sample microchamber. It is designed to accommodate the fluid sample in size similar to the sample microchamber but also will have space for additional reaction components.
  • the reaction microchamber contains the predetermined antigen or a plurality of predetermined antigens and an antigen accelerator. In the case of the second test of the invention, it only contains the antigen accelerator and is antigen free.
  • predetermined antigen is an antigen that the fluid sample is to be tested in order to determine if the subject providing the sample has had a previous exposure to the antigen. Any antigen that produces a leucocyte measurement factor can be such predetermined antigen and selected by the tester who wishes to test the subject for exposure to the predetermined antigen.
  • Bioterrorist type antigens for anthrax, botulism, the plague, smallpox, tularemia, hemorrhagic fever, brucellosis, glanders, melioidosis, psittacisis, Q fever, typhus, encephalitis, toxins, afflatoxins, nipah, hantavirus, west Nile fever, hendra and rift valley fever are notable test antigens of high consequence.
  • Other disease states and antigens of lower consequence including but not limited to, tetanus, diphtheria, and monslia, chemicals known to activate leucocytes, tuberculosis, and the like may also be tested.
  • African horse sickness African swine fever, akabane virus, avian influenza, bluetongue, bovine spongiform, swine fever, coccidioidomycosis, bovine pleuropneumonia, caprine pleuropneumonia, foot and mouth disease, goat and sheep pox, heartwater lumpy skin disease, catarrhal fever, menangle virus, Newcastle disease, peste despooinants, rinderpest, screwworm myiasis, swine vesicular disease, vesicular stomatitis, and the like can be tested.
  • a subject can be exposed to either a low consequence antigen, that is one who's result to the subject is either low impact or recoverable or exposed to high consequence antigens such as bioterrorist agents that result in serious injury or death.
  • Another source of antigens are those antigens purposely introduced into a subject by way of vaccination and the determination of the efficacy of the vaccination is desired, e.g. vaccines against the flu, vaccines against bio-terror agents or vaccines against cancer antigens.
  • a sample of the antigen proportional to the microvolume of body fluid is placed in the first reaction microchamber. It can be as a liquid or in a dry powder or coating of the microchamber or by other method known for placing materials in micro type chambers.
  • the "antigen accelerator” is also placed in the first reaction microchamber as well as the control second reaction microchamber.
  • the antigen accelerator is a composition which stimulates, amplifies and or accelerates the production of measurement factors in leucocyte upon exposure to an antigen. These compositions are known in the art and are being further developed. For example, ⁇ 2 -macroglobulin is known as an antigen accelerator ( Vaccine 2001, 20, 554-562 ).
  • the antigen accelerator is placed in the reaction microchambers as a liquid or powder or coating similarly to the antigen. Where the antigen accelerator needs to be in contact with the antigen as is the case with most known accelerators, the antigen accelerator can coat the predetermined antigen or antigens and the resulting mixture used in the reaction chamber.
  • reaction time of the invention will be the time the leucocyte in the fluid need in the presence of the antigen and accelerator to develop measurement factors.
  • the presences of the antigen accelerator is designed to produce, in such microvolumes, very quick reactions times. In other words, the measurement factors would develop an antigen reaction optimally in about an hour or two.
  • the exact time will vary plus or minus depending on the predetermined antigen but one skilled in the art can determine that exact time for the predetermined antigen in the presence of the antigen accelerator. In the case of multiple predetermined antigens being added to the reaction microchamber, the predetermined time would be the longest predetermined time of each of the predetermined antigen reaction times separately.
  • the reaction microchambers and observation microchambers can be the same microchambers when one is not using a colorant that is cytotoxic. Transportation is previously described and can be accomplished simply by opening a valve between the reaction and observation microchambers and allowing capillary action to move the liquid. It is necessary during this process to mix the reacted fluid with one or more leucocyte observation colorant.
  • the leucocyte observation colorant can be placed in the channel between microchambers or can be placed in the observation microchamber itself or both. This can be done by dry coating either the channel or microchamber.
  • Leucocyte observation colorants are compounds known to differentially color morphological factors, in a leucocyte and cause various colorations (spectral factors) at various wavelengths based on the leucocytes reaction or lack of reaction to the antigen.
  • Examples of such colorants include but are not limited to: Astrozone Orange, also known as Basic Orange 21 which is 3-trimethyl-2(2-(2-methyl-1H-indol-3yl)-vinyl)-3H-indolium chloride.
  • cytotoxic colorants include Acridine Orange, Ethidium Bromide, Griefswalder's Blue, Blue Borrel, Rhodanile Blue, Toluidine Blue, Night Blue, Prune Pure, Hofmann's Violet, Basic Red 13, Basic Violet 16, Carbocyanine K-5, and mixtures of above. Many of the colorants are cytotoxic. When selecting a cytotoxic colorant in the present invention it is required that it is only allowed to be in contact with cells the minimum time. In the embodiment of the invention where the observation and reaction chambers are separate the minimum time in contact is achieved. Where the predetermined time is short enough or the colorant is not cytotoxic the embodiment where the reaction and observation microchambers of each test are the same microchamber can be used.
  • the "observation microchamber” is a chamber for which leucocytes measurement factors can easily be observed by optical scan.
  • the microchamber is designed to spread out the fluid sample in such a manner to make a field by field, XYZ scan possible. See, for example, US patent 6,350,613 which describes such a microchamber and optical scan thereby.
  • the observation microchamber can also be the reaction microchamber.
  • the fluid can be optically scanned for leucocyte measurement factors based on color produced by various morphological and spectral factors by the leucocyte observation colorant.
  • This is done in one embodiment using multi-wavelength fluorescence image analysis as described in US patent 6,350,613 .
  • a method is described for differentiating different cells or cell subtypes by observation of the fluorescing cell at two or more wavelengths.
  • One could also use an optical microscope or any other manual or automatic method or device for observing fluorescence i.e. observing or scanning the samples. It has been discovered by the inventors hereof, that in addition to cell subtypes, measurement factors of leucocytes can be determined using the described method of the present invention.
  • Morphological factor changes can easily be determined by the scan picking up the different physical characteristics highlighted by the colorant and noting that they represent normal leucocyte or antigen reacted leucocyte.
  • spectral factor differences can be measured, the differences between normal and antigen reacted leucocytes can also be compared.
  • Fluorescence image analysis is now widely carried out and cell based machines and hand microscopes as well as ones that digitize and analyze information are readily available.
  • Companies such as Universal imaging, for example, make machines that can easily be adapted by one skilled in the art for use with the method and device of the invention. In the alternative, these machines could easily be modified to accept the device of the invention and automatically perform the desired scan.
  • Image analysis software is in general use and can be used to create digital pixels of the image viewed by the scan and assign intensity and color numbers for each pixel. The digital data can then be used in not only finding morphological factors it can be used in a number of ways to determine spectral factors.
  • Measurement factors include both morphological factors and spectral factors.
  • Morphological factors include shape, diameter, area, volume and perimeter of the cell, area and volume of nucleus, shape of nucleus, percentage of total volume occupied by nucleus, eccentricity of nucleus, etc.
  • Spectral factors include sum of all the pixel intensities that make up the total cell area at any of 16 different emission wavelengths, ratios of each sum to any other sum, average pixel intensity at each of emission wavelength, coefficient of variation of the pixel intensities at each of 16 wavelengths, sum of the total pixels in the area identified as cytoplasm (not nucleus), ratios of each sum to any other sum in the cytoplasm, average pixel intensity at each wavelength of cytoplasm pixels, coefficient of variation of the cytoplasm pixels at each of 16 wavelengths, geometric center of light emitted by all pixels, etc
  • Measurement factors can also be quantified using measured morphological and spectral factors measured by multi-wave fluorescence image analysis and measuring the number or intensity of reacted leucocytes exhibiting the measurement factors as compared to the control test of the invention.
  • Image analysis of multi-wave fluorescence information converts picture information into a series of digital information as pixels of differing color and intensity. This information may be stored and compared in a computer, pixel by pixel. By comparing the control test scan to the antigen scan and comparing the change of both color and relative intensity gives the process of the invention in addition to the qualitative information, determining if the leucocyte is responding to the antigen, it gives a quantitative result by the number and intensity of pixels corresponding to the intensity of the antigen response of the responding leucocyte. The greater the number and intensity of pixels, the greater the response and therefore the greater the production of presensitized leucocyte for the given antigen.
  • One skilled in the art can measure the measurement factors using the color variations and/or other colorimetric measuring factors known in the art as well as the image analysis by multi-wavelength fluorescence. By looking for leucocyte changes based on cell-mediated immune response rather than antibody production the method of the invention is able to quickly and accurately provide the objects of the invention.
  • the second test of the invention is identical to the first test except the second reaction microchamber is antigen free.
  • all the factors of the second test are identical except for exposure to the antigen.
  • the digital data may be compared to the first test data. By comparing the differences between the two tests it can be seen statistically and visually when there is a difference in measurement factors of the leucocyte sample of the patient. These differences are an indicator of a positive preexposure to the predetermined antigen.
  • the device is made in a disposable format.
  • This device would be made of plastic or other inexpensive disposable material.
  • the device of the invention containing the subject sample can be discarded in an appropriate manner and the tester need never come in contact with the contents.
  • the disposable microfluidic device can be constructed credit card size more or less similar to other microfluidic assays such that it fits in a reader portion of an image analyzer that can read the colorimetric data from the tests by either moving the test device around or moving a reader in the analyzer or both to take readings of the type in the above referenced patents and also described herein.
  • Figure 1 is a plan view of the microfluidic device of the invention where the reaction chambers and observation chambers are separate.
  • the leucocyte-antigen assay microfluidic device 2 has a length and width similar to other standard disposable microfluidic assay tests of its type. So a 2 in by 4 inch size or other size as needed to fit the analyzer being used and fit the microchambers as described herein is used. As shown in this embodiment the microfluidic device 2 is rectilinear however any convenient shape can be employed that would allow the tests to be placed thereon.
  • the device 2 is fitted with a sealed cover 3 which covers the entire top of the device 2 except for the sample microchambers 10 and 11 that remain uncovered to allow a sample of fluid to be placed therein.
  • the size of the microchambers is as described elsewhere herein but in general allows for a drop or two of fluid from a test subject.
  • sample microchamber 11 is for the first test and sample microchamber 10 is for the second test (i.e. the control test).
  • the shape of these microchambers is not critical but should be a size that accommodates the means being used to add the fluid sample whether by dropper or dropped from a lanceted finger or the like.
  • Fluid channels 23 and 21 flow from their respective sample microchambers 10 and 11 and deliver the fluid sample to reaction microchambers 12 and 13 respectively.
  • These reaction microchambers 12 and 13 are roughly the same size as the sample microchambers. Since in this embodiment capillary action will be moving the sample it is anticipated that some fluid will remain in each of the microchambers shown as well as the channels.
  • the reaction chamber 13 is coated with both a predetermined antigen/ antigen accelerator complex 40, that in this embodiment coats the bottom of reaction chamber 13.
  • Reaction chamber 12 is coated with only the antigen accelerator 41.
  • Fluid channels 22 and 24 deliver the fluid sample to observation microchambers 15 and 14 respectively. Interposed in each of channels 22 and 24 are microvalves 30.
  • Each microvalve 30 can block the capillary flow from a reaction chamber to prevent flow into the observation chambers 14 and 15 until the predetermined time described above has elapsed.
  • Observation microchambers 14 and 15 have a different geometry than the other 4 microchambers in this embodiment.
  • Observation microchambers 14 and 15 have a wedge shape tapering from a widest point where the fluid channels 22 and 24 enter the microchambers to the far end of the observation microchambers 14 and 15 where the thickness is roughly the thickness of a single blood cell or about 3 microns.
  • Disposed in fluid channels 22 and 24 are leucocyte observation colorants 45.
  • Figure 2 to is a plan perspective wherein the first and second sample microchambers are the same microchamber and wherein each of the respective first reaction microchamber and first observation microchamber; and second reaction microchamber and second observation microchamber are the same.
  • This embodiment of the invention is useful where the colorant is not cytotoxic or where the predetermined time is so short as to not cause cytotoxic problems.
  • microfluidic device 5 can be fitted with cover 6 which covers the entire top 7 of device 5 except for the opening for sample chamber 20.
  • the joint first and second sample microchamber 20 is the chamber for adding the body fluid sample to be assayed.
  • Two fluid channels 21 and 22 for the first and second test respectively are in fluid communication with sample chamber 20.
  • microchambers 17 and 16 are wedge shaped similar to the observation chambers 14 and 15 of Figure 1 .
  • microchamber 17 contains the antigen-antigen accelerator complex as well as the leucocyte observation colorant 45.
  • Latent Johne's Disease infection in dairy cattle Johnes is the second costliest disease in the dairy industry and it is caused by the Mycobacterium avium paratuberculosis organism (MAP).
  • MAP Mycobacterium avium paratuberculosis organism
  • the organism is known to be one of the slowest growers in the tuberculosis family, is extremely difficult to detect in latent asymptomatic infections, and it generates practically no antibody response in dairy cattle for months, or even years.
  • MAP upon initial exposure to cattle, MAP triggers an initial response in a subpopulation of bovine leukocytes (memory T-cells). The record of that initial encounter is retained in the immunological memory of the T Cell..
  • a bovine whole blood sample that been previously exposed to MAP is placed in the sample microchamber of the microfluidic device of the invention.
  • the blood is then moved to the first reaction microchamber and re-stimulated using protein antigens that are unique to MAP, in combination with the antigen accelerator ⁇ 2 -macroglobulin.
  • the sample is moved to the observation microchamber and the sample is treated with the colorant, Astrozone Orange which had previously been placed in the observation microchamber.
  • a second test is run with the blood identical to the first with the only difference being the absence of antigen in the reaction microchamber.
  • the level of T cell response is quantified scanning the cells in the observation microchamber of both the first and second test and counting the number of T Cells that show a response to the presence of the unique antigen as evidenced by a marked change in the morphological and spectral factors in the first test as compared to the cells in the second test that does not contain the MAP antigens.

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Claims (21)

  1. Ein Verfahren eines Leukozytenantigen-vermittelten mikrofluidischen Assays, ausgeführt an einer mikrofluidischen Vorrichtung zum Testen einer Leukozyten-enthaltenden Körperflüssigkeit eines Subjekts, um es vorher einem vorbestimmten Antigen auszusetzen, das dazu führt, die Leukozyten bezüglich des Antigens vorzusensibilisieren, das Verfahren weist dabei Folgendes auf:
    die Ausführung eines ersten Tests, der wiederum folgende Schritte aufweist:
    a) die Einfüllung eines Mikrovolumens einer ersten Probe der Leukozyten-enthaltenden Körperflüssigkeit des Subjekts in eine erste Proben-Mikrokammer;
    b) den Transport über die Kapillarwirkung oder durch den Einsatz einer Mikropumpe der ersten Körperflüssigkeitsprobe zu einer ersten Reaktions-Mikrokammer, die ein vorbestimmtes Antigen und einen Antigen-Beschleuniger enthält, und es diesen ermöglicht, über eine vorbestimmte Zeit miteinander zu reagieren, wobei der Antigen-Beschleuniger eine Zusammensetzung ist, die die Herstellung von Messfaktoren in Leukozyten, wenn sie einem Antigen ausgesetzt werden, stimuliert, verstärkt und/oder beschleunigt;
    c) den Transport über die Kapillarwirkung oder durch den Einsatz einer Mikropumpe der reagierten ersten Körperflüssigkeitsprobe zu einer ersten Beobachtungs-Mikrokammer;
    d) die Mischung eines oder mehrerer Leukozyten-Beobachtungs-Färbemittel mit der reagierten ersten Körperflüssigkeitsprobe während des Transports zur Beobachtungs-Mikrokammer oder in der Beobachtungs-Mikrokammer;
    e) die Ausführung eines optischen Scans der reagierten Körperflüssigkeitsprobe in der ersten Beobachtungs-Mikrokammer unter Bedingungen, die die Messung mindestens eines Messfaktors der Leukozyten in der reagierten ersten Körperflüssigkeitsprobe erlauben, wobei der Messfaktor oder die Faktoren, die gemessen werden, morphologische Faktoren, Spektralfaktoren oder beides sind;
    die Ausführung eines zweiten Tests, der die folgenden Schritte aufweist:
    a) die Einfüllung eines Mikrovolumens einer zweiten Probe der Leukozyten-enthaltenden Körperflüssigkeit des Subjekts in eine zweite Proben-Mikrokammer;
    b) den Transport über die Kapillarwirkung oder durch den Einsatz einer Mikropumpe der zweiten Körperflüssigkeitsprobe zu einer antigenfreien zweiten Reaktions-Mikrokammer, die denselben Antigen-Beschleuniger enthält wie im ersten Test, und es diesen ermöglicht, über eine vorbestimmte Zeit miteinander zu reagieren;
    c) den Transport über die Kapillarwirkung oder durch den Einsatz einer Mikropumpe der zweiten Körperflüssigkeitsprobe zu einer zweiten Beobachtungs-Mikrokammer;
    d) die Mischung desselben Leukozyten-Beobachtungs-Färbemittels wie im ersten Test mit der reagierten zweiten Körperflüssigkeitsprobe während des Transports zur zweiten Beobachtungs-Mikrokammer oder in der zweiten Beobachtungs-Mikrokammer;
    e) die Ausführung eines optischen Scans der reagierten zweiten Körperflüssigkeitsprobe in der zweiten Beobachtungs-Mikrokammer, unter Bedingungen, die die Messung derselben Leukozyten-Messfaktoren wie im ersten Test erlauben;
    den Vergleich der Ergebnisse des ersten Test-Scans mit dem zweiten Test-Scan, um zu bestimmen, ob Differenzen zwischen den beiden Scans eine Reaktion der Leukozyten mit dem Antigen anzeigen, aufgrund der Tatsache, dass die Leukozyten, dadurch, dass sie dem Antigen zuvor ausgesetzt waren, bezüglich dieses Antigens vorsensibilisiert waren,
    wobei optional die erste Reaktions-Mikrokammer und die erste Beobachtungs-Mikrokammer dieselbe Mikrokammer sind und die zweite Reaktions-Mikrokammer und die zweite Beobachtungs-Mikrokammer dieselbe Mikrokammer sind,
    und wobei entweder (a) das Färbemittel nicht zytotoxisch ist oder (b) das Färbemittel ein zytotoxisches Färbemittel ist, aber unter dem Vorbehalt, dass wenn die erste Reaktions-Mikrokammer und die erste Beobachtungs-Mikrokammer dieselbe Mikrokammer sind und/oder wenn die zweite Reaktions-Mikrokammer und die zweite Beobachtungs-Mikrokammer dieselbe Mikrokammer sind, das besagte Färbemittel nur die Mindestzeit in Kontakt mit den Zellen sein darf.
  2. Eine mikrofluidische Vorrichtung für die Verwendung bei der Ausführung des Verfahrens eines Leukozytenantigen-vermittelten mikrofluidischen Assays gemäß Anspruch 1, wobei die Vorrichtung Folgendes aufweist:
    a) eine erste Flüssigkeitsproben-Beimischungs-Mikrokammer in Fluidverbindung, über einen ersten Fluidkanal, mit einer ersten Proben-Reaktions-Mikrokammer, die ein vorbestimmtes Antigen und einen Antigen-Beschleuniger enthält, wobei der Antigen-Beschleuniger eine Zusammensetzung ist, die die Herstellung von Messfaktoren in Leukozyten, wenn sie einem Antigen ausgesetzt werden, stimuliert, verstärkt und/oder beschleunigt;
    b) eine erste Proben-Beobachtungs-Mikrokammer in Fluidverbindung, über einen zweiten Fluidkanal, mit der ersten Proben-Reaktions-Mikrokammer;
    c) ein oder mehrere Leukozyten-Beobachtungs-Färbemittel, die entweder im zweiten Fluidkanal, der ersten Beobachtungs-Mikrokammer oder in beidem positioniert sind;
    d) eine zweite Flüssigkeitsproben-Beimischungs-Mikrokammer in Fluidverbindung, über einen dritten Fluidkanal, mit einer antigenfreien zweiten Proben-Reaktions-Mikrokammer, die denselben Antigen-Beschleuniger enthält wie die erste Proben-Reaktions-Kammer;
    e) eine zweite Proben-Beobachtungs-Mikrokammer in Fluidverbindung, über einen vierten Fluidkanal, mit der zweiten Proben-Reaktions-Mikrokammer;
    f) dieselben Leukozyten-Beobachtungs-Färbemittel wie im ersten Test, die entweder im vierten Fluidkanal, der zweiten Beobachtungs-Mikrokammer oder beidem positioniert sind;
    g) eine Vorrichtung zur Weiterleitung, über die Kapillarwirkung oder den Einsatz einer Mikropumpe, mindestens eines Teils jeder Flüssigkeitsprobe durch ihren jeweiligen Fluidkanal in nachfolgende Mikrokammern,
    wobei optional die erste Proben-Beimischungs-Mikrokammer und die zweite Proben-Beimischungs-Mikrokammer dieselbe Mikrokammer sind,
    und wobei optional die erste Reaktions-Mikrokammer und die erste Beobachtungs-Mikrokammer dieselbe Mikrokammer sind und die zweite Reaktions-Mikrokammer und die zweite Beobachtungs-Mikrokammer dieselbe Mikrokammer sind,
    und wobei entweder (a) das Färbemittel nicht zytotoxisch ist oder (b) das Färbemittel ein zytotoxisches Färbemittel ist, aber unter dem Vorbehalt, dass wenn die erste Reaktions-Mikrokammer und die erste Beobachtungs-Mikrokammer dieselbe Mikrokammer sind und/oder wenn die zweite Reaktions-Mikrokammer und die zweite Beobachtungs-Mikrokammer dieselbe Mikrokammer sind, das besagte Färbemittel nur die Mindestzeit in Kontakt mit den Zellen sein darf.
  3. Ein Verfahren gemäß Anspruch 1 oder eine Vorrichtung gemäß Anspruch 2, wobei das Mikrovolumen der ersten Probe und das Mikrovolumen der zweiten Probe jeweils 100 Mikroliter oder weniger beträgt.
  4. Ein Verfahren gemäß Anspruch 1 oder eine Vorrichtung gemäß Anspruch 2, wobei die Beobachtungs-Mikrokammern jeweils so gestaltet sind, dass sie die Flüssigkeitsprobe in der Weise ausbreiten, dass ein feldweiser XYZ-Scan möglich ist.
  5. Ein Verfahren gemäß Anspruch 1 oder eine Vorrichtung gemäß Anspruch 2, wobei das Färbemittel nicht zytotoxisch ist und die erste Reaktions-Mikrokammer und die erste Beobachtungs-Mikrokammer dieselbe Mikrokammer sind und die zweite Reaktions-Mikrokammer und die zweite Beobachtungs-Mikrokammer dieselbe Mikrokammer sind,
  6. Ein Verfahren gemäß Anspruch 1, wobei die Messfaktoren, die gemessen werden, (a) morphologische Faktoren oder (b) sowohl morphologische Faktoren als auch Spektralfaktoren sind.
  7. Ein Verfahren gemäß Anspruch 6, wobei die morphologischen Faktoren ausgewählt sind aus Form, Durchmesser, Bereich, Volumen und Umfang der Zelle, Bereich und Volumen des Zellkerns, der Form des Zellkerns, des Prozentsatzes des Gesamtvolumens, das vom Zellkern eingenommen wird, und der Exzentrizität des Zellkerns.
  8. Ein Verfahren gemäß Anspruch 1, wobei die Messungen in ihrer Art sowohl qualitativ als auch quantitativ sind.
  9. Ein Verfahren gemäß Anspruch 1, wobei die Anzahl der reagierenden Zellen, die einen Messfaktor zeigen, beobachtet wird, genauso wie die Plus- oder Minus-Intensität der Differenz zwischen den beiden Tests.
  10. Ein Verfahren gemäß Anspruch 1, wobei mindestens drei Messfaktoren verglichen werden.
  11. Ein Verfahren gemäß Anspruch 1, wobei der optische Scan durch eine Mehrfach-Wellenlängen-Fluoreszenzbildanalyse oder eine Vorrichtung gemäß Anspruch 2 erreicht wird, wobei die erste und die zweite Beobachtungs-Mikrokammer so gestaltet sind, dass sie mit der Mehrfach-Wellenlängen-Fluoreszenzbildanalyse abgelesen werden können.
  12. Ein Verfahren gemäß Anspruch 1, wobei das Verfahren den Vergleich des zweiten Test-Scans mit dem ersten Test-Scan aufweist, und den Vergleich sowohl der Farbe als auch der relativen Intensität.
  13. Ein Verfahren gemäß Anspruch 12, wobei das Verfahren ein quantitatives Ergebnis aus der Anzahl und Intensität der Pixel liefert, das der Intensität der Antigen-Reaktion auf die reagierende Leukozyte entspricht, ebenso eine qualitative Information, die anzeigt, ob die Leukozyte auf das Antigen reagiert.
  14. Ein Verfahren gemäß Anspruch 1, wobei die quantitative Antigen-Leukozyten-Reaktivität entweder von der Messung der Pixelanzahl, der relativen Intensität der Pixel, hinsichtlich der Fluoreszenz der reaktiven Leukozyten des Subjekts, oder von beidem bestimmt wird, wenn man die Ergebnisse des ersten Scans mit denen des zweiten vergleicht.
  15. Ein Verfahren gemäß Anspruch 1, wobei die Leukozyten-enthaltende Flüssigkeit des Subjekts zu einem bestimmten Grad verdünnt wird, bevor sie in den Tests verwendet wird.
  16. Ein Verfahren gemäß Anspruch 1, wobei sowohl die Schritte des ersten Tests und die Schritte des zweiten Tests mit Vollblut ausgeführt werden.
  17. Ein Verfahren gemäß Anspruch 1, wobei das gesamte Verfahren mit einer Einwegvorrichtung oder einer Vorrichtung gemäß Anspruch 2 ausgeführt wird, die eine Einwegvorrichtung ist.
  18. Eine Vorrichtung gemäß Anspruch 2, wobei mindestens einer der Kanäle ein Ventil hat, das darin untergebracht ist, um die Flüssigkeitsströmung zwischen den Mikrokammern zu regeln.
  19. Ein Verfahren gemäß Anspruch 1 oder eine Vorrichtung gemäß Anspruch 2, wobei der Antigen-Beschleuniger α2-Makroglobulin ist.
  20. Ein Verfahren gemäß Anspruch 1 oder eine Vorrichtung gemäß Anspruch 2, wobei das Leukozyten-Beobachtungs-Färbemittel ein trockenes metachromatisches Einfärbemittel ist.
  21. Ein Teilesatz, der eine mikrofluide Vorrichtung gemäß Anspruch 2 aufweist, und ein Apparat, der einen optischen Mehrfeld-Scan in den Beobachtungs-Mikrokammern in der mikrofluiden Vorrichtung ausführen kann.
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CA2565981C (en) 2015-02-17
US20150275163A1 (en) 2015-10-01
US20090011451A1 (en) 2009-01-08
EP1751268A2 (de) 2007-02-14
NZ552034A (en) 2009-11-27
EP1751268A4 (de) 2011-01-26
US10620203B2 (en) 2020-04-14
WO2006085897A2 (en) 2006-08-17
AU2005327290A1 (en) 2006-08-17
IL179115A (en) 2013-07-31
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